515 Horm. Metab. Res. 7 (1975) 515-520
© Georg Thieme Verlag Stuttgart
A Human Placental Hormone (UTPH) with Uterine Growth and DNA Promoting Effects* F. Beas** A. Salinas, F. Gonzalez, C. Teran*** and P. Szendro***, with the technical assistance of M. Figueroa and L. Aguilera Laboratorio de Investigaciones Pediatricas. Departamento Materno Infantil. Facultad de Medicina, Sede Sur, Universidad de Chile, Santiago, Chile
A human placental protein previously described is studied in order to expand its biological characterization. Dose-response-curve of its action on uterine growth of prepubcral mice showed to be a significant logarithmic function and this effect is neulralized by its specific antiserum. It is also demonstraled that the uterine growth-promoting effecI is not due either to estrogen, protein-bound estrogen, progesterone or chorionic gonadolrophin. Experimental evidence is given to demonstrate that the mechanism of action of this placental protein is 10 stimulate the synthesis and 10 increase the concentration of DNA in uterine lissue, differing thcn in Ihis respect from the effect of estrogen. Previous work has shown that this placental protein is present in full-term placcntas within similar ranges as HCG and HPL. It is also detectcd in blood during pregnancy and acts biologically in at least two target organs: uterus and mammary gland.
ty of this placental protein, namely its effect on the growth of the uterus. Materials and Methods The method used in the isolation of the placental protein under study has already been described (Beas and Flores 1969). Briefly Ihis consists of a placental extraction with 0.1 N acetic acid and subsequent gel filtration in a column of Sephadex G-75. The biological effect of the placental protein on the weight of the uterus was determined by the method of Klinefelter (1943). Prepuberal, 21 day old femalc mice of the Balb strain were used. The animals were injected intraperitoneally at 12 hours intervals with three doses of the placental protein and then sacrificed 72 hours after the first injection. The uteri were carefully dissected and immediately weighed on a precision microtorque balance with ± 1 mg of accuracy.
The method for preparation of antisera has been described elsewhere (Beas and Gonztilez 1968). An hydrolyzate of the Therefore Ihe name of uterotrophic placental protein (UTPH) placental protein was prepared according to the method of has been suggested for this substance. Beling (1963).
Key-Words:Human Placental Protein - Human Placental Hormone- Uterotrophic Placental Hormone (UTPHJ Uterus - DNA - Uterine Growth and DNA.
Progesterone determinations were performed by radioimmunoassay by the Bio-Science Laboratories, California, U.S.A.
The effect of the placental protein on the incorporation of labeled Cl4-formate into protein, DNA and RNA of the mice uterus was studied as folIows: 21 day old prepuberal Introduction fe male mice were fasted for 24 hours; water was a1lowed ad The endocrine function of the placenta has been ac- Iibitum. 300 Ilg of placental protein dissolved in 0.3 nii of cepted for many years. This consists of the synthesis 0.9% NaCI was injected intraperitoneally afterward. For comstudies, animals of the same strain and age under of steroids, mainly estrogens and gestagens as weil as parative the same experimental conditions were injected with 10 Ilg peptidic hormones such as chorionic gonadotrophin of 17-beta-estradiol in 0.3 ml of saline wilh 1% ethanol and placental lactogen (Simmer 1968). In the course (v/v). Control animals were injected with saline and 1% ethanol. All the animals were given intraperitoneally 81lCi of of our studies which were directed toward improving of the method for isolating placentallactogen from hu- Cl4-forrnate in 0.3 ml of saline 2 hours bcfore they were sacrificed. The animals were killed by asphyxiation with gas man term placentas, a placental protein was obtained 2, 4 and 6 hours following the first injection and the uteri which differed in its physicochemical and biological were removed immediately. For each determination, 3 uteri characteristics from all other hormones described up were pooled at randorn within each group. The organs were rapidly stripped of fat, weighed, placed with physiological until now. The methods for the preparation of this solution in a Potter-Eveljhem glass~lass apparatus and homoprotein as weil as its physicochemical characteristics, genized to a final volume of 2 ml. This and all subsequent have been previously described elsewhere (Beas and procedures were carried out at 0-2 0 C. After removing aliquots Gonzalez 1968; Beas and Flores 1968, 1969, Beas for protein determination (Lowry et al., 1951), DNA and RNA were determined by the method of Schmidt and Tann· 1972). This paper adds to Dur preliminary report (Beas and Flores 1968, 1969) on the biological activi- hauser (1945) modified by Fleck and Munro (1962) and Blobel and Potter (1968). Uteri were homogenized and precipitated with perchloric acid (PCA) to a final concentration • This work was partly supported by Comision Nacional of 0.3 N and then washed once with the same acid by centride Investigacion Cientffica y Tecnologica de Chile and fugation. A precipitate was obtained and it was hydrolyzed Comision de Investigacion Cientifica y Creacion Artlstica with 0.3 N potassium hydroxide at 37 0 C for one hour and Universidad de Chile. subsequently extracted with PCA 10 a final concentration of •• Present address: Casilla 5370, Santiago 3, Chile. 0.3 N to hydrolyze the RNA in the supernatant. The precipit••• Present address: Max Planck Institute, Wilhelmshaven, ate resuIting from this extraction with PCA was again washGermany. Received: 15 Feb. 1975 Accepted: 30 Aug. 1975
Downloaded by: Universite Laval. Copyrighted material.
Summary
516
F. Beas, A. Salinas, F. Gonziles, C. Teran and P. Szendro , • 256.32 log. -43407 r .0.SJ73t p
© Georg Thieme Verlag Stuttgart
A Human Placental Hormone (UTPH) with Uterine Growth and DNA Promoting Effects* F. Beas** A. Salinas, F. Gonzalez, C. Teran*** and P. Szendro***, with the technical assistance of M. Figueroa and L. Aguilera Laboratorio de Investigaciones Pediatricas. Departamento Materno Infantil. Facultad de Medicina, Sede Sur, Universidad de Chile, Santiago, Chile
A human placental protein previously described is studied in order to expand its biological characterization. Dose-response-curve of its action on uterine growth of prepubcral mice showed to be a significant logarithmic function and this effect is neulralized by its specific antiserum. It is also demonstraled that the uterine growth-promoting effecI is not due either to estrogen, protein-bound estrogen, progesterone or chorionic gonadolrophin. Experimental evidence is given to demonstrate that the mechanism of action of this placental protein is 10 stimulate the synthesis and 10 increase the concentration of DNA in uterine lissue, differing thcn in Ihis respect from the effect of estrogen. Previous work has shown that this placental protein is present in full-term placcntas within similar ranges as HCG and HPL. It is also detectcd in blood during pregnancy and acts biologically in at least two target organs: uterus and mammary gland.
ty of this placental protein, namely its effect on the growth of the uterus. Materials and Methods The method used in the isolation of the placental protein under study has already been described (Beas and Flores 1969). Briefly Ihis consists of a placental extraction with 0.1 N acetic acid and subsequent gel filtration in a column of Sephadex G-75. The biological effect of the placental protein on the weight of the uterus was determined by the method of Klinefelter (1943). Prepuberal, 21 day old femalc mice of the Balb strain were used. The animals were injected intraperitoneally at 12 hours intervals with three doses of the placental protein and then sacrificed 72 hours after the first injection. The uteri were carefully dissected and immediately weighed on a precision microtorque balance with ± 1 mg of accuracy.
The method for preparation of antisera has been described elsewhere (Beas and Gonztilez 1968). An hydrolyzate of the Therefore Ihe name of uterotrophic placental protein (UTPH) placental protein was prepared according to the method of has been suggested for this substance. Beling (1963).
Key-Words:Human Placental Protein - Human Placental Hormone- Uterotrophic Placental Hormone (UTPHJ Uterus - DNA - Uterine Growth and DNA.
Progesterone determinations were performed by radioimmunoassay by the Bio-Science Laboratories, California, U.S.A.
The effect of the placental protein on the incorporation of labeled Cl4-formate into protein, DNA and RNA of the mice uterus was studied as folIows: 21 day old prepuberal Introduction fe male mice were fasted for 24 hours; water was a1lowed ad The endocrine function of the placenta has been ac- Iibitum. 300 Ilg of placental protein dissolved in 0.3 nii of cepted for many years. This consists of the synthesis 0.9% NaCI was injected intraperitoneally afterward. For comstudies, animals of the same strain and age under of steroids, mainly estrogens and gestagens as weil as parative the same experimental conditions were injected with 10 Ilg peptidic hormones such as chorionic gonadotrophin of 17-beta-estradiol in 0.3 ml of saline wilh 1% ethanol and placental lactogen (Simmer 1968). In the course (v/v). Control animals were injected with saline and 1% ethanol. All the animals were given intraperitoneally 81lCi of of our studies which were directed toward improving of the method for isolating placentallactogen from hu- Cl4-forrnate in 0.3 ml of saline 2 hours bcfore they were sacrificed. The animals were killed by asphyxiation with gas man term placentas, a placental protein was obtained 2, 4 and 6 hours following the first injection and the uteri which differed in its physicochemical and biological were removed immediately. For each determination, 3 uteri characteristics from all other hormones described up were pooled at randorn within each group. The organs were rapidly stripped of fat, weighed, placed with physiological until now. The methods for the preparation of this solution in a Potter-Eveljhem glass~lass apparatus and homoprotein as weil as its physicochemical characteristics, genized to a final volume of 2 ml. This and all subsequent have been previously described elsewhere (Beas and procedures were carried out at 0-2 0 C. After removing aliquots Gonzalez 1968; Beas and Flores 1968, 1969, Beas for protein determination (Lowry et al., 1951), DNA and RNA were determined by the method of Schmidt and Tann· 1972). This paper adds to Dur preliminary report (Beas and Flores 1968, 1969) on the biological activi- hauser (1945) modified by Fleck and Munro (1962) and Blobel and Potter (1968). Uteri were homogenized and precipitated with perchloric acid (PCA) to a final concentration • This work was partly supported by Comision Nacional of 0.3 N and then washed once with the same acid by centride Investigacion Cientffica y Tecnologica de Chile and fugation. A precipitate was obtained and it was hydrolyzed Comision de Investigacion Cientifica y Creacion Artlstica with 0.3 N potassium hydroxide at 37 0 C for one hour and Universidad de Chile. subsequently extracted with PCA 10 a final concentration of •• Present address: Casilla 5370, Santiago 3, Chile. 0.3 N to hydrolyze the RNA in the supernatant. The precipit••• Present address: Max Planck Institute, Wilhelmshaven, ate resuIting from this extraction with PCA was again washGermany. Received: 15 Feb. 1975 Accepted: 30 Aug. 1975
Downloaded by: Universite Laval. Copyrighted material.
Summary
516
F. Beas, A. Salinas, F. Gonziles, C. Teran and P. Szendro , • 256.32 log. -43407 r .0.SJ73t p
